Beta-lactamase enzymes represent a major mechanism of resistance among bacteria to beta-lactam antibiotics, which include penicillins, cephalosporins and carbapenems. These enzymes catalyse the irreversible hydrolysis of the amide bond of the beta-lactam ring to create ineffective antimicrobial agents. On the basis of molecular structure classification and catalytic mechanisms the beta-lactamases can be divided into four classes: A, B, C, and D. Classes A, C and D are serine enzymes and comprise the majority of the beta-lactamases (Ambler, 1980). These enzymes generally inactivate penicillins or cephalosporins and often show a preference for one of these two antibiotics.
Class B beta-lactamases are metallo-enzymes that require one or two zinc ions as a cofactor for enzyme activity. The metallo-beta-lactamases constitute group 3 in the Bush-Jacoby-Madeiros functional classification (Bush, 1998). This schema is primarily based on substrate profiles, their sensitivity to EDTA and their resistance to serine beta-lactamase inhibitors. Based on structural similarities in the region that coordinates zinc binding, metallo-beta-lactamases can be divided into three subgroups, B1, B2, and B3 (Galleni et al., 2001). Subgroup B1 possesses three histidines and one cysteine as the key zinc coordinating residues. Crystallographic structures have been described for many subgroup B1 enzymes such as BcII of Bacillus cereus (Carfi et al, 1995 and 1998a), CcrA of Bacteroides fragilis and (Carfi et al., 1998b) and IMP-1 of Pseudomonas aeruginosa (Concha et al., 2000). Correspondingly, subgroup B2 lactamases have an arginine residue, instead of histidine, at the first position of the principal zinc binding motif, NXHXD (SEQ ID NO:39). Recently, the first crystal structure of a subgroup B2 enzyme (CphA) has been solved by Garau et al. (2005). Subgroup B3 contains enzymes with multimeric structure (Walsh et al., 2005).
Metallo-beta-lactamases show a broad spectrum substrate profile including penicillins and cephalosporins, and they are resistant to the action of common conventional serine beta-lactamase inhibitors such as clavulanic acid sulbactam and tazobactam. Furthermore, unlike most of the serine beta-lactamases, metallo-beta-lactamases have the capability to hydrolyze carbapenems such as meropenem and imipenem. Various numbers of bacteria are known to produce metallo-beta-lactamases. They are commonly expressed among the Enterobacteriae ogenus (including Serratia marcescens, Klebsiella pneumoniae, Citrobacter freudii, Shigella flexneri), Pseudomonas aeruginosa, Stenobacterium maltophila, Acinetobacter genus, Bacteroides fragilis, Bacillus cereus, Flavobacteruim odoratum, and Bacteroides fragilis (Walsh et al., 2005).
Beta-lactamases can be utilized as pharmaceutical proteins to inactivate unabsorbed beta-lactams in the gastro intestinal tract in order to prevent the beta-lactam induced adverse effects including alterations in intestinal normal microbiota and the overgrowth of beta-lactam resistant bacteria (WO93/13795, WO2004/016248). For efficient beta-lactamase therapy in the small intestinal tract the enzyme should be resistant to the action of intestinal proteases in the presence of bile acids and preserve high enzymatic activity at a wide range of pH (5.5-7.5).
The feasibility of targeted enzyme therapy in canine and mouse models was demonstrated by employing a Bacillus licheniformis serine beta-lactamase during parenteral ampicillin medication (Harmoinen et al., 2004, Mentula et al., 2004, Stiefel et al., 2003). However, the substrate profile of this enzyme essentially limits its use as a drug substance since it has poor capacity to hydrolyze cephalosporins, carbapenems or penicillins in the presence of beta-lactamase inhibitors. Consequently, a new protease resistant beta-lactamase enzyme with broad beta-lactam spectrum is indispensable to extend the use of beta-lactamase therapy among hospitalized patients under intravenous medication with various beta-lactams.
Metallo-beta-lactamases are known to inactivate various types of beta-lactams and they are resistant to inhibitors of serine beta-lactamases. Bacillus cereus strains are known to produce metallo-beta-lactamase that belongs to group B1. A semi purified recombinantly produced metallo-beta-lactamase sample of a clinical Bacillus cereus 98ME1552 isolate was shown to eliminate the overgrowth of potential pathogenic bacteria in a mouse model (Stiefel et al., 2005). However, the present inventors found that this metallo-beta-lactamase preparation contained a mixture of beta-lactamase variants, which declines its value as a pharmaceutical protein, since variations of a drug substance reduce the robustness of the production process, increase batch to batch variations, and make clinical trials difficult, which of course has a negative impact on its registration as a medicament.
The present invention now provides means for reducing the amino terminal heterogenicity that was found to be associated with recombinant production of metallo-beta-lactamase. The invention further provides modified metallo-beta-lactamases that can be produced in substantially pure form and that can be used in the manufacture of pharmaceutical compositions.